POS0151 SINGLE-CELL RNA SEQUENCING OF BRONCHOALVEOLAR LAVAGE FLUID AND BLOOD REVEALS DISEASE-SPECIFIC CHARACTERISTICS OF IMMUNE CELLS IN CONNECTIVE TISSUE DISEASE-ASSOCIATED INTERSTITIAL LUNG DISEASE PATIENTS
Authors: A. Hirano, W. Fujii, A. Sakashita, K. Baßler, M. Kadoya, A. Omoto, W. Fukuda, T. Seno, M. Wada, M. Kohno, Y. Kawahito
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Connective tissue disease-associated interstitial lung disease (CTD-ILD) is a serious complication and an important prognostic factor of various autoimmune diseases. Many cell types and both innate and adaptive immune systems are implicated in the pathogenesis of fibrotic lung injury. The presentation and clinical course of CTD-ILD are diverse, and the heterogeneity reflects differences in underlying pathogenic mechanisms. <h3>Objectives</h3> The objective of this study is to reveal the different characteristics of immune cells among diseases. <h3>Methods</h3> We collected bronchoalveolar lavage fluid (BALF) and peripheral blood from patients who newly complicated interstitial lung disease and agreed to participate in this study; 5 patients with Sjögren syndrome (SS), 8 patients with dermatomyositis (DM), 6 patients with rheumatoid arthritis (RA), 7 patients with systemic sclerosis (SSc), 4 patients with ANCA-associated vasculitis (AAV), and 12 patients with idiopathic interstitial pneumonia (IIP) included. We applied Seq-Well, a portable platform of single-cell RNA sequencing<sup>[1]</sup>, to analyze gene expressions in immune cells in BALF and blood. We compared the distribution of immune cells and differential gene expressions in BALF and blood among diseases. <h3>Results</h3> We compared immune cell numbers in BALF among diseases. We found more T cells in patients with DM and SS, more B cells in patients with SS, more neutrophils in patients with RA, and more alveolar macrophages (AMs) in patients with AAV or SSc. We further performed gene ontology enrichment analysis of each immune cells in BALF. In patients with DM, the gene expression pathways related to the response to virus was increased in T cells. On the other hand, in patients with SS, the gene expression pathways involved in innate immunity and antigen presentation were enhanced in B cells. In addition, the gene expression associated with T cell activation was enhanced in T cells. In neutrophils from patients with RA, the gene expression involved in inflammation was increased. We next performed gene ontology enrichment analysis of blood. In monocytes and neutrophils, patients with DM had increased gene expression pathways associated with viruses, while patients with SS had increased gene expression of both innate immune pathways and acquired immunity. These results suggested that the common pathways were enhanced in BALF lymphocytes and in blood monocytes and neutrophils in patients with DM and SS. <h3>Conclusion</h3> Our comprehensive single-cell analysis of BALF and blood from CTD-ILD patients has revealed that the phenotypes and states of immune cells differ among diseases (Figure 1). We found that the common pathways were enhanced in BALF and blood, i.e., pathways associated with response to virus were enhanced in DM while innate immunity and antigen presentation pathways were enhanced in SS, suggesting the relationship between systemic immune abnormality and ILD. These results may lead to the optimal diagnosis and the precision medicine for patients with CTD-ILDs. <h3>Reference</h3> [1]Gierahn TM et al, Seq-Well: portable, low-cost RNA sequencing of single cells at high throughput. Nat Methods 2017;14(4):395-398. <h3>Acknowledgements:</h3> NIL. <h3>Disclosure of Interests</h3> Aiko Hirano Speakers bureau: Sanofi K.K., Wataru Fujii Speakers bureau: Boehringer Ingelheim Japan, Inc., Mitsubishi Tanabe Pharma Corporation, Grant/research support from: Boehringer Ingelheim Japan, Inc., GlaxoSmithKline K.K., Takeda Pharmaceutical Co.,Ltd., Aki Sakashita: None declared, Kevin Baßler: None declared, Masatoshi Kadoya: None declared, Atsushi Omoto Speakers bureau: AbbVie GK, Chugai Pharmaceutical Co. Ltd., Ono Pharmaceutical Co., Ltd., Daiichi-Sankyo Inc., Astellas Pharma Inc., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Asahi Kasei Pharma Corp, Novartis Pharma KK, Gilead Sciences Inc., Janssen Pharmaceutical K.K., GlaxoSmithKline K.K, Eli Lilly Japan K.K, Wataru Fukuda Speakers bureau: Chugai Pharmaceutical Co., Eisai Co., Ltd., Eli Lilly Japan K.K., AbbVie GK, Ono Pharmaceutical Co. Ltd., Astellas Pharma Inc., UCB Japan Co. Ltd., Pfizer Japan Inc., Giread Science K.K., Asahikasei Pharma Co., Daiichi-Sankyo Inc., Takahiro Seno Speakers bureau: Asahi Kasei Pharma, Janssen Pharmaceutical K.K., Boehringer Ingelheim Japan, Inc, Makoto Wada Speakers bureau: AbbVie GK, Astellas Pharma Inc., Bristol Myers Squibb Co., Chugai Pharmaceutical Co. Ltd., Eisai Co. Ltd., Eli Lilly Japan K.K, Janssen Pharmaceutical K.K., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Sanofi Japan K.K., Masataka Kohno Speakers bureau: AbbVie GK, Astellas Pharma Inc., Bristol Myers Squibb Co., Chugai Pharmaceutical Co. Ltd., Daiichi Sankyo Co., Eli Lilly Japan K.K, Janssen Pharmaceutical K.K., Mitsubishi Tanabe Pharma Co., Asahikasei Pharma Co., Pfizer Japan Inc., Sanofi Japan K.K., Boehringer Ingelheim Co. Ltd., UCB Japan Co. Ltd., Eisai Co. Ltd., GlaxoSmithKline K.K., Yutaka Kawahito Speakers bureau: AbbVie GK, Asahi Kasei Pharma Corp, Astellas Pharma Inc., Ayumi Pharmaceutical Corp., Boehringer Ingelheim Japan Inc., Bristol Myers Squibb Co., Chugai Pharmaceutical Co. Ltd., Daiichi Sankyo Co., Eisai Co. Ltd., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Takeda Pharmaceutical Co. Ltd., Teijin Pharma Ltd., UCB Japan Co. Ltd., GlaxoSmithKline K.K., Eli Lilly Japan K.K, Janssen Pharmaceutical K.K., Sanofi Japan K.K., Gilead Sciences Inc., Mylan EPD, Grant/research support from: AbbVie GK, Asahi Kasei Pharma Corp, Astellas Pharma Inc., Ayumi Pharmaceutical Corp., Boehringer Ingelheim Japan Inc., Bristol Myers Squibb Co., Chugai Pharmaceutical Co. Ltd., Daiichi Sankyo Co., Eisai Co. Ltd., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Takeda Pharmaceutical Co. Ltd., Teijin Pharma Ltd., GlaxoSmithKline K.K., Gilead Sciences Inc.